The reaction of physiological significance catalyzed by the carbonic anhydrases is the hydration of carbon dioxide: CO2 + H2O yields or reversible action HCO3 + H+. This catalysis requires attack on CO2 by zinc-bound hydroxide followed by proton shuttle, to regenerate the zinc- bound hydroxide. The unifying goal of this proposal is to understand the catalytic mechanism of the carbonic anhydrases with emphasis on the rate- limiting proton transfer steps. A concurrent goal is to apply Marcus rate theory both to understand the catalytic mechanism of carbonic anhydrase and to elucidate the significance of the parameters of the Marcus theory to proton transfer in an enzyme. We plan to use site-specific mutagenesis and chemical modification to vary the location in the active-site cavity of the intramolecular proton shuttle and the pK of the zinc-bound water. For this purpose we have expression systems for three isozymes of carbonic anhydrase (II, III, and V) giving us a thousand-fold range of catalytic activities in the wild-type enzymes and a range and geometries for study. For each of these isozymes a crystal structure is available, and further structural implications of strategic mutations will be determined by x-ray crystallography. Stopped-flow and 18 O exchange between CO2 and water measured by mass spectrometry will be used to obtain rate constants for intramolecular proton transfer. Solvent hydrogen isotope effects will be measured to determine the rate-limiting nature of the proton transfer. We will evaluate the relationship between the basicity of the zinc-bound water and the rate of its nucleophilic attack on CO2 in carbonic anhydrase and mutants. We will apply Marcus rate theory to determine and interpret the intrinsic energy barriers for proton transfer and relate them to the structural and chemical features of the active site of carbonic anhydrase. The overall usefulness of the marcus theory to understanding proton transfer in carbonic anhydrase will be evaluated.